The present invention relates to plants, especially transgenic plants, plant parts and plant cells overproducing an iron binding protein (e.g. ferritin) and having an enhanced resistance against a wide range of abiotic and biotic oxidative stress conditions (e.g. against treatment with paraquat or fusaric acid and against viral, bacterial and fungal infections). The invention also comprises nucleic acid sequences encoding an alfalfa ferritin or functional variants thereof and the use of said sequences for rendering plants resistant against oxidative stress conditions.
The invention is useful for reducing environmental damages of crops caused by a wide variety of stress conditions.
With respect to the present specification and claims, we will use the following technical terms in accordance with the given definitions. With regard to the interpretation of the present invention, it shall be understood that the below defined terms are used in accordance with the given definitions even if said definitions might not be in perfect harmony with the usual interpretation of said technical term.
A xe2x80x9cfunctional variantxe2x80x9d of a protein is a polypeptide the amino acid sequence of which can be derived from the amino acid sequence of the original protein by the substitution, deletion and/or addition of one or more amino acid residue in a way that, in spite of the change in the amino acid sequence, the functional variant retains at least a part of at least one of the biological activities of the original protein that is detectable for a person skilled in the art. A functional variant is generally at least 50% homologous (i.e. the amino acid sequence of it is 50% identical), advantageously at least 70% homologous and even more advantageously at least 90% homologous to the protein from which it can be derived. Any functional part of a protein or a variant thereof is also termed functional variant.
The term xe2x80x9coverproducingxe2x80x9d is used herein in the most general sense possible. A special type of molecule (usually a polypeptide or an RNA) is said to be xe2x80x9coverproducedxe2x80x9d in a cell if it is produced at a level significantly and detectably higher (e.g. 20% higher) than natural level. Overproduction of a molecule in a cell can be achieved via both traditional mutation and selection techniques and genetic manipulation methods. The term xe2x80x9cectopic expressionxe2x80x9d is used herein to designate a special realisation of overproduction in the sense that, for example, an ectopically expressed protein is produced at a spatial point of a plant where it is naturally not at all (or not detectably) expressed, that is, said protein is overproduced at said point.
A plant, plant part, a plant tissue or a plant cell is said to have an xe2x80x9cenhanced resistancexe2x80x9d against a damaging effect, eg. damaging agent, if it can tolerate a significantly and detectably (e.g. at least 20%) stronger damaging effect, eg. dose or intensity of damaging agent, of the same type, without suffering any detectable damage, than its natural counterpart would do.
Within the framework of the present description a xe2x80x9cferritin proteinxe2x80x9d is defined, as it is usual in the art, as a protein capable binding iron ions (Theil E. C., 1987, Ann. Rev. Biochem. 56: 289-315). The members of the eucariotic ferritin family are highly conserved both in their amino acid sequence and three dimensional structure (Lobreaux, S. et al., 1992, Biochem. J. 288: 931-939).
The term xe2x80x9coxidative stressxe2x80x9d is again used in very general sense comprising all kind of abiotic (e.g. treatment with different chemical agents or exposure to extreme weather conditions like high or low temperature or drought) and biotic (infection by different infectious agents) stress conditions in the manifestation of damaging effects of which oxidatively induced active radicals play a detectable role.
During their different developmental stages plants are exposed to an extremely wide range of both biotic and abiotic stress conditions. It is, thus, a very important task of high economic significance to develop new breeding stocks of enhanced general stress resistance.
Under stress conditions such as high light intensity, UV-B irradiation, heavy metal contamination, high or low temperature, water deficiency, flooding, wounding, infection by viruses, bacteria, fungi, damage caused by insects and the like, oxygen toxicity can significantly contribute to the damage of crop plants. Reactive oxygen species as singlet oxygen, superoxide radical (O2), hydroxyl radical (OH+) and hydrogen peroxide (H2O2) play a key role in injury of stressed plants. There is good evidence that the biological damage attributed to superoxide and hydrogen peroxide is dependent on the presence of iron. The intracellular pool of free iron can react with H2O2 or O2xe2x88x92 giving rise to the very reactive hydroxyl radical via Haber-Wiess or Fenton reaction (Halliwell and Gutteridge, 1984, Biochem. J. 219: 1-14). Intracellularly, most of the non-metabolised iron is sequestered in ferritin; therefore ferritin is able to restrict the availability of iron and so the generation of the very reactive hydroxyl radicals. cDNAs encoding ferritin have been isolated from variety of plant species. These proteins are highly conserved both in amino acids sequence and three dimension structure (Lobreoux S. et al. 1992, Biochem. J. 288: 931-939.). Ferritins are localised in chloroplasts and iron can activate their synthesis (Seckbach, J. 1982, J. Plant. Nutr. 5: 369-394; Lobreaux et al. 1992, Plant Mol. Biol. 19: 563-575). Under normal growth conditions ferritin is synthesised only in embryo and not in vegetative organs, like roots and leaves (Lobreaux and Briat: Biochem. J. 1991, 274: 601-606).
Significant antioxidant effect of ferritin molecules can be expected in systems where ferritin synthesis and degradation is released from the normal metabolic regulation. It has been demonstrated that during oxidative stress conditions, degradation of ferritin molecules occurs and the so released iron ions highly accelerate the production rate of the damaging radical species (Cairo et al. 1995, Journal of Biochemical Chemistry 270: 700-703). Numerous traditional plant breeding and genetic manipulation approaches are known in the art for improving the resistance of specific crops against preselected desired stress conditions (e.g. against cold, drought, UV light or pathogens). These known methods, however, will not be individually detailed herein as they are all highly different from the approach of the invention. The common feature of all these previously disclosed approaches is that they enhance the resistance of different plants against a single preselected stress condition (or against a limited groups of stress conditions of the same origin) e.g. by expressing a specific resistance gene. However, no specific approach providing plants with resistance against a wide range of both abiotic and biotic stress conditions is known in the art.
It is, thus, an object of the present invention to provide a novel and general method suitable to provide crops, especially transgenic crops, with enhanced resistance against a wide range of both abiotic and biotic stress conditions.
According to another aspect, it is also an object of the invention to provide crops and breeding material, advantageously transgenic, having increased resistance against a wide range of both abiotic and biotic stress conditions.
On the basis of the foregoing disclosure it has, thus, became clear to the inventors that a substantially new genetic manipulation approach is to be developed so as to achieve the above defined objects, possibly targeting a common step in the damaging mechanism of the different abiotic and biotic stress conditions.
The approach of the present invention is, thus, based on the novel theoretical concept that overproducing or ectopically expressing ferritin or other iron binding proteins, e.g. transferring, in different organs of plants will lower the intracellular iron concentration and, therefore, reduce the damaging effects of oxygen induced free radicals. It is important to emphasise hereby that the approach of the invention is absolutely novel, as there is no method disclosed in the art that would provide plants with resistance against a wide range of both abiotic and biotic stress conditions. Furthermore, there is no approach disclosed so far according to which a ferritin protein is overproduced in plants for any reason in any manner.
Therefore, to achieve the above-defined objects of the invention we have cloned a ferritin cDNA gene from alfalfa (Medicago sativa L.) and overproduced it in vegetative tissues of tobacco plants.
It was found, that in accordance with the basic concept of the invention, the transgenic tobacco plants expressing the alfalfa ferritin ectopically in their vegetative tissues show significantly higher resistance towards both abiotic (treatment with different chemicals) and biotic (viral, bacterial and fungal infections) stress conditions than the starting tobacco plants and transformants show improved general adaptation and regeneration characteristics, as well.
It should be also emphasised that according to the basic concept of the invention it is probable that overproduced ferritin molecules express their general protective effect via binding free iron ions in the plant cells, it can be assumed that by overproducing other iron binding proteins, such as transferring, a similar protective effect could be achieved.
The present invention, therefore, provides plant cells overproducing an iron binding protein and having an enhanced resistance against oxidative stress. The plants of the invention are advantageously produced by genetic manipulation methods known per se but can also be produced via traditional mutation and selection techniques. The transformed plants of the invention show significantly higher resistance to oxidative stress conditions than their unmodified counterparts not expressing elevated levels of an iron binding protein.
The plant cells according to the invention are advantageously transgenic cells transformed by the introduction of a nucleic acid, eg in the form of vector, coding for the expression of an iron binding protein, advantageously ferritin.
According to preferred embodiments of the invention there are provided plant cells of the invention are overproducing a ferritin having the amino acid sequence of SEQ ID No 2 as shown in the attached sequence listing, or a functional variant thereof, said functional variant being advantageously at least 50%, more advantageously at least 70% and even more advantageously at least 90% homologous to said ferritin polypeptide.
The invention further provides plants, advantageously transgenic plants, and parts thereof comprising cells according to the invention.
According to a preferred embodiment of the invention, the intensity of the photosynthetic reactions is not decreased following a 70 hours treatment with 10 xcexcM paraquat in the leaves of the plants of the invention.
Plant, plant parts or plant cells according to the invention advantageously have an enhanced resistance against fusaric acid treatment and/or infections of viral and/or bacterial and/or fungal origin.
The invention also provides isolated, enriched, cell free and/or recombinant nucleic acids of sequence comprising or consisting of a nucleotide sequence coding for an alfalfa ferritin having the sequence of SEQ ID No: 2, as shown in the sequence listing included herewith, and functional variants thereof. Preferably the nucleic acids have homology of at least 90% with that of SEQ ID No 1 (the DNA sequence in FIG. 1), more preferably at least 95% and most preferably at least 98%. Preferably these nucleic acids are cDNAs, recombinant vectors or other recombinant constructs.
The invention also comprises the use of these nucleic acids for the preparation of plant cells, plant parts and plants according to the invention.
The present disclosure and examples below demonstrate that synthesis of the iron binding protein ferritin in vegetative tissues of plants provides resistance against paraquat generated free radicals. In agreement with this observation we have also demonstrated a significant reduction of the symptoms after infection of the transgenic plants of the invention with a wide range of unrelated pathogens. This novel technology according to the present invention based on ectopic expression of an iron binding protein, therefore, potentially has high agronomic significance in that it may reduce primary oxidative damage in crops caused by either biotic or abiotic stress conditions. Though not wishing to be bound to any theoretical interpretation, we think that the above findings support the assumption that iron ions are more effectively bound in the plants according to the invention as a consequence of the overproduction of an iron binding protein (ferritin) and, therefore, damage caused by oxygen induced free radicals are reduced and general adaptation and regenerative properties of the plants of the invention are improved. Plants according to the invention will also be able to show resistance against other stress conditions not tested so far, e.g. against extremely low or high temperatures and drought, where such iron mediated degeneration is implicated.
Though it is considered to be quite clear from the above disclosure for a person skilled in the art, we also wish to emphasise here the universality of the approach according to the invention. We have demonstrated that ectopical expression of an iron binding protein of alfalfa origin in vegetative tissues confers resistance to transgenic tobacco plants against a wide variety of stress conditions. As the pioneering approach of the invention is based on the absolutely general concept of reducing the concentration of iron ions in the targeted tissues of plants, a person skilled in the art will understand that the advantages of the invention can not be restricted to the specific embodiments shown but this novel approach for ensuring general stress resistance to plants can be used in the case of any other crops of agronomic or horticultural significance.
It is also demonstrated that the ectopic overexpression of the embryo specific alfalfa ferritin in vegetative tissues of tobacco plants, using two different types of promoters (one of plant and the one of viral origin) is not at all damaging to the targeted tissue. It should, thus, be contemplated that any type of plant specific promoters (either constitutive or spatially and/or developmentally regulated) can be used in the invention for the overexpression of an iron binding protein in the desired plant part or tissue.
As the concentration of harmful radicals is usually the highest in the photosynthesising green tissues, these tissues are the first useful targets of the approach according to the invention. It should be understood, however, that, because of its above demonstrated highly universal basic concept, the invention is not at all limited to conferring stress resistance to green tissues, but it is also useful for making resistant all other plant parts of interest (e.g. root, stem, flower, fruit or specific parts of the foregoing) supposedly exposed to any kind of oxidative stress conditions (e.g. to infection by any type of pathogens).